This lab can be chaotic since essentially two labs are running at once: half the students will be in the TC facility splitting cells and the other half will be in the main lab running a gel. Halfway through they will switch.

In pre-run about 2ug of pCX-del3 was cut for each and recovered 30ul of 0.05ug/ul cut DNA after gel purification

(5ul in 500 to measure conc. IOD260 = 50ug/ml

It will be easiest to dilute all DNAs to approx same concentration (pCX-EGFP and pCX-del5 to 0.25ug/ul and all pCX-del3s to 0.05ug/ul would be ideal). Be sure to use STERILE WATER and TC pipets... in Spring of 04 there was terrible contamination of these transfections.

Prep Work
For today's lab need 24 well dish (1 per pair) in media WITHOUT antibiotics

The day will be a repetitive one at the FACS facility. Each group with come with 16 samples to collect. Print out two copies of data for each group, one for them and one for the lab's notes. MES cell culture is done after today!! w00t

Materials required:

TC reagents

PBS (20 mL per group)

Trypsin (4 mL per group)

OptiMEM (2 mL per group)

FACS tubes

16 per group, plus extras

BD Falcon tubes only, VWR cat # 60819-310 (1000x) or 60819-295 (500x)

Day of Lab:

No quiz.

TA will run flow cytometer, and faculty will guide student preparations in the lab, or vice-versa.